Nonhomologous end joining: A good solution for bad ends

Double strand breaks pose unique problems for DNA repair, especially when broken ends possess complex structures that interfere with standard DNA transactions. Nonhomologous end joining can use multiple strategies to solve these problems. It further uses sophisticated means to ensure the strategy chosen provides the ideal balance of flexibility and accuracy

The fidelity of the ligation step determines how ends are resolved during nonhomologous end joining

Nonhomologous end joining (NHEJ) can effectively resolve chromosome breaks despite diverse end structures, but it is unclear how the steps employed for resolution are determined. We sought to address this question by analyzing cellular NHEJ of ends with systematically mispaired and damaged termini. We show NHEJ is uniquely proficient at bypassing subtle terminal mispairs and radiomimetic damage by direct ligation. Nevertheless, bypass ability varies widely, with increases in mispair severity gradually reducing bypass products from 85% to 6%. End-processing by nucleases and polymerases is increased to compensate, though paths with the fewest number of steps to generate a substrate suitable for ligation are favored. Thus, both the frequency and nature of end processing are tailored to meet the needs of the ligation step. We propose a model where the ligase organizes all steps during NHEJ within the stable paired-end complex to limit end processing and associated errors

Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

Nonhomologous end-joining (NHEJ) is the main pathway for repair of DNA double-strand breaks (DSBs), the most cytotoxic form of DNA damage resulting from ionizing radiation, chemotherapeutics, and normal cellular processes. The mechanisms that control NHEJ play key roles in development, in immunity, and in response to cancer therapy; however, the current state of knowledge regarding the physical nature of the NHEJ repair process is limited. Here we used super-resolution microscopy to define the organization of NHEJ complexes in cells, showing that long filaments form at either side of the break. Single-molecule FRET revealed dynamic behavior in which breaks can pair in an adjacent, non–end-to-end configuration

A semiquantitative metric for evaluating clinical actionability of incidental or secondary findings from genome-scale sequencing

As genome-scale sequencing is increasingly applied in clinical scenarios, a wide variety of genomic findings will be discovered as secondary or incidental findings, and there is debate about how they should be handled. The clinical actionability of such findings varies, necessitating standardized frameworks for a priori decision making about their analysis

Evaluating the Clinical Validity of Gene-Disease Associations: An Evidence-Based Framework Developed by the Clinical Genome Resource

Supplemental Data Supplemental Data include 65 figures and can be found with this article online at http://dx.doi.org/10.1016/j.ajhg.2017.04.015. Supplemental Data Document S1. Figures S1–S65 Download Document S2. Article plus Supplemental Data Download Web Resources ClinGen, https://www.clinicalgenome.org/ ClinGen Gene Curation, https://www.clinicalgenome.org/working-groups/gene-curation/ ClinGen Gene Curation SOP, https://www.clinicalgenome.org/working-groups/gene-curation/projects-initiatives/gene-disease-clinical-validity-sop/ ClinGen Knowledge Base, https://search.clinicalgenome.org/kb/agents/sign_up OMIM, http://www.omim.org/ Orphanet, http://www.orpha.net/consor/cgi-bin/index.php With advances in genomic sequencing technology, the number of reported gene-disease relationships has rapidly expanded. However, the evidence supporting these claims varies widely, confounding accurate evaluation of genomic variation in a clinical setting. Despite the critical need to differentiate clinically valid relationships from less well-substantiated relationships, standard guidelines for such evaluation do not currently exist. The NIH-funded Clinical Genome Resource (ClinGen) has developed a framework to define and evaluate the clinical validity of gene-disease pairs across a variety of Mendelian disorders. In this manuscript we describe a proposed framework to evaluate relevant genetic and experimental evidence supporting or contradicting a gene-disease relationship and the subsequent validation of this framework using a set of representative gene-disease pairs. The framework provides a semiquantitative measurement for the strength of evidence of a gene-disease relationship that correlates to a qualitative classification: “Definitive,” “Strong,” “Moderate,” “Limited,” “No Reported Evidence,” or “Conflicting Evidence.” Within the ClinGen structure, classifications derived with this framework are reviewed and confirmed or adjusted based on clinical expertise of appropriate disease experts. Detailed guidance for utilizing this framework and access to the curation interface is available on our website. This evidence-based, systematic method to assess the strength of gene-disease relationships will facilitate more knowledgeable utilization of genomic variants in clinical and research settings

Performance of ACMG-AMP Variant-Interpretation Guidelines among Nine Laboratories in the Clinical Sequencing Exploratory Research Consortium

Evaluating the pathogenicity of a variant is challenging given the plethora of types of genetic evidence that laboratories consider. Deciding how to weigh each type of evidence is difficult, and standards have been needed. In 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published guidelines for the assessment of variants in genes associated with Mendelian diseases. Nine molecular diagnostic laboratories involved in the Clinical Sequencing Exploratory Research (CSER) consortium piloted these guidelines on 99 variants spanning all categories (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign). Nine variants were distributed to all laboratories, and the remaining 90 were evaluated by three laboratories. The laboratories classified each variant by using both the laboratory's own method and the ACMG-AMP criteria. The agreement between the two methods used within laboratories was high (K-alpha = 0.91) with 79% concordance. However, there was only 34% concordance for either classification system across laboratories. After consensus discussions and detailed review of the ACMG-AMP criteria, concordance increased to 71%. Causes of initial discordance in ACMG-AMP classifications were identified, and recommendations on clarification and increased specification of the ACMG-AMP criteria were made. In summary, although an initial pilot of the ACMG-AMP guidelines did not lead to increased concordance in variant interpretation, comparing variant interpretations to identify differences and having a common framework to facilitate resolution of those differences were beneficial for improving agreement, allowing iterative movement toward increased reporting consistency for variants in genes associated with monogenic disease

Clinical Sequencing Exploratory Research Consortium: Accelerating Evidence-Based Practice of Genomic Medicine

Despite rapid technical progress and demonstrable effectiveness for some types of diagnosis and therapy, much remains to be learned about clinical genome and exome sequencing (CGES) and its role within the practice of medicine. The Clinical Sequencing Exploratory Research (CSER) consortium includes 18 extramural research projects, one National Human Genome Research Institute (NHGRI) intramural project, and a coordinating center funded by the NHGRI and National Cancer Institute. The consortium is exploring analytic and clinical validity and utility, as well as the ethical, legal, and social implications of sequencing via multidisciplinary approaches; it has thus far recruited 5,577 participants across a spectrum of symptomatic and healthy children and adults by utilizing both germline and cancer sequencing. The CSER consortium is analyzing data and creating publically available procedures and tools related to participant preferences and consent, variant classification, disclosure and management of primary and secondary findings, health outcomes, and integration with electronic health records. Future research directions will refine measures of clinical utility of CGES in both germline and somatic testing, evaluate the use of CGES for screening in healthy individuals, explore the penetrance of pathogenic variants through extensive phenotyping, reduce discordances in public databases of genes and variants, examine social and ethnic disparities in the provision of genomics services, explore regulatory issues, and estimate the value and downstream costs of sequencing. The CSER consortium has established a shared community of research sites by using diverse approaches to pursue the evidence-based development of best practices in genomic medicine

Which came first: validity or clinical testing? The example of long QT genes

Aim: To investigate the potential relationship between the strength of evidence for a gene-disease association and inclusion of the gene on a targeted, indication-based gene panel test for hereditary long QT syndrome (LQTS) and to explore factors that may influence laboratory decisions about the inclusion or exclusion of genes from these clinical tests.Methods: A comprehensive literature search was performed to quantify existing evidence supporting putative LQTS gene-disease associations. This evidence included the year that the gene was first implicated in LQTS, the total number of published cases of LQTS attributed to the gene, and the presence of published segregation and functional data for the gene. To explore the possible relationship between the published evidence for clinical validity of each gene and availability of clinical genetic testing, semi-structured interviews were conducted with key laboratory stakeholders. Representatives from nine US laboratories offering clinical LQTS gene testing agreed to be interviewed regarding decision-making about when and why genes comprising their clinical LQTS test offerings were added.Results: Genes associated with LQTS before 2006 generally had more reported cases of LQTS and the greatest amount of supporting segregation and functional data prior to being offered as a clinical test. For genes first linked to LQTS after 2006, these trends are less linear and the timeframe between initial report and inclusion on clinical test menus decreased substantially. Advances in technology, lifting of patents, clinician request, and literature searches were cited as the main factors that influence composition of LQTS gene panel tests. Paradoxically, one lab director noted that it may require more evidence to remove a gene than to add a gene to a clinical test panel.Conclusion: Our evaluation of the LQTS genes illustrates the nuanced relationship between published evidence supporting a gene-disease association and availability of clinical testing. Expert assessment of clinical validity of gene-disease associations may help laboratories to determine gene panel content. The ultimate impact of such information on the composition of clinical gene tests as well as their utilization by clinicians and coverage by health insurance policies remains to be seen

Requirements for 5′dRP/AP lyase activity in Ku

The non-homologous end joining (NHEJ) pathway is used in diverse species to repair chromosome breaks, and is defined in part by a requirement for Ku. We previously demonstrated mammalian Ku has intrinsic 5′ deoxyribosephosphate (5′dRP) and apurinic/apyrimidinic (AP) lyase activity, and showed this activity is important for excising abasic site damage from ends. Here we employ systematic mutagenesis to clarify the protein requirements for this activity. We identify lysine 31 in the 70 kD subunit (Ku70 K31) as the primary candidate nucleophile required for catalysis, but additional mutation of Ku70 K160 and six other lysines within Ku80 were required to eliminate all activity. Ku from Saccharomyces cerevisiae also possesses 5′dRP/AP lyase activity, and robust activity was also reliant on lysines in Ku70 analogous to K31 and K160. By comparison, these lysines are not conserved in Xenopus laevis Ku, and Ku from this species has negligible activity. A role for residues flanking Ku70 K31 in expanding the range of abasic site contexts that can be used as substrate was also identified. Our results suggest an active site well located to provide the substrate specificity required for its biological role